Reversible Immunosensor for the Continuous Monitoring of Cortisol in Blood Plasma Sampled with Microdialysis

Cortisol is a steroid hormone involved in a wide range of medical conditions. The level of the hormone fluctuates over time, but with traditional laboratory-based assays, such dynamics cannot be monitored in real time. Here, a reversible cortisol sensor is reported that allows continuous monitoring of cortisol in blood plasma using sampling by microdialysis. The sensor is based on measuring single-molecule binding and unbinding events of tethered particles. The particles are functionalized with antibodies and the substrate with cortisol-analogues, causing binding and unbinding events to occur between particles and substrate. The frequency of binding events is reduced when cortisol is present in the solution as it blocks the binding sites of the antibodies. The sensor responds to cortisol in the high nanomolar to low micromolar range and can monitor cortisol concentrations over multiple hours. Results are shown for cortisol monitoring in filtered and in microdialysis-sampled human blood plasma.

H ormones are regulatory molecules that are transported through the body to control processes such as metabolism, growth and development, inflammation, emotions and mood, reproductive function, and sleep. 1−4 Cortisol is a steroid stress hormone that fluctuates strongly and affects almost all tissues and organs in the body. Under healthy conditions, cortisol concentrations have a circadian profile 4−7 with high concentrations in the morning (0.1−0.7 μM) 4−6 and low concentrations during the night (0.1−0.4 μM). 4,6 Elevated cortisol levels (above 0.7 μM) can result from chronic stress and relate to conditions such as heart disease, obesity, burnout, and Cushing's syndrome. 1,8,9 Monitoring cortisol−time profiles in individuals could aid in perioperative patient care and in the diagnosis and treatment of conditions with dysregulated or irregular cortisol levels such as Cushing's syndrome. 1,5,10,11 Sensors based on lateral-flow assays 12 and electrochemical detection 13 have been reported for the measurement of cortisol in biological fluids such as sweat, saliva, plasma, and serum. 7,12−19 Cortisol−time profiles were recorded using separate sensors for every individual sample. 20 In some studies, multiple samples were measured on a single sensor, as the cortisol concentration increased as a function of time. 21,22 However, for measuring arbitrary cortisol−time profiles, a fully reversible sensor is needed that can record fluctuating cortisol concentrations with phases of increasing as well as decreasing cortisol concentration as a function of time.
A recently developed continuous biosensing technology called biosensing by particle mobility (BPM) is based on measuring reversible interactions between biofunctionalized particles and a biofunctionalized substrate. 23−29 Previous BPM studies reported the monitoring of ssDNA, thrombin, and creatinine. Here, a BPM competition assay sensor is demonstrated for the monitoring of cortisol, using cortisolanalogues and anti-cortisol antibodies. We report measurements with multiple cycles of increasing and decreasing cortisol concentrations. Data are shown for cortisol in buffer, cortisol in filtered blood plasma, and cortisol sampled from blood plasma using a microdialysis catheter probe, as this represents an interfacing technology that is suitable for future patient monitoring. ■ RESULTS

BPM Competition Immunoassay for Continuous
Cortisol Monitoring. This study aims to develop a biosensor for continuous cortisol monitoring in plasma, using a BPM competition immunoassay (Figure 1). In BPM, thousands of particles ( Figure S1B) are tracked simultaneously, temporal reversible molecular bonds form between particles and substrate, and these induce observable changes in the mobility of the particles ( Figure S1C). The average frequency of bond formation, recorded as the switching activity, relates to the analyte concentration in solution and is used as the signal to monitor the cortisol concentration as a function of time. The particles are functionalized with anti-cortisol antibodies and the substrate with cortisol analogue. In the absence of cortisol, the switching frequency is high as the particle repeatedly binds to and unbinds from the substrate. An increasing cortisol concentration in solution leads to a gradual decrease of the switching frequency of the particles (Figure 2A), as the occupation of antibodies by cortisol from solution lowers the probability that antibodies bind to the cortisol analogues on the substrate. ). The sensor response shows the expected behavior of a BPM competition assay, i.e., the activity signal is inversely proportional to the  analyte concentration. In a BPM competition assay, the binding between the antibody and analyte-analogue should be strong enough to cause bound states of the particles that have a sufficiently long lifetime for reliable state detection. However, the binding should not be too strong because long bound-state lifetimes result in low switching activity. In addition, the affinity between the antibody and analyte should be high enough so that low analyte concentrations can be detected. Yet, the affinity should be low enough to permit reversible binding and enable the monitoring of increases as well as decreases of the analyte concentration as a function of time. After comparing different antibodies, we selected one antibody and optimized the densities of the antibody on particles and analyte-analogue on the substrate to achieve single-molecule interactions (see SI 2). This resulted in activity signals in the range of tens of mHz and sensitivity to cortisol concentrations in the range between 100 nM and 10 μM, as shown in Figure 2.
The sensor demonstrates high similarity between consecutively measured dose−response curves, allowing the monitoring of fluctuating cortisol concentrations over a period of 5 h. The measured blank signals at 0, 170, and 300 min are similar within about 10%, demonstrating the stability of the sensor.
The observed time of the sensor depends on the concentration step change. In the measurement shown in Figure 2, a decrease in concentration from 30 to 0 μM shows a characteristic time of about 15 min (see the dashed gray curve), while an increase in concentration from 0 to 30 μM gives a response time of about 5 min (visible in the experiment as a rapid signal change between the last gray data point and the first green data point, at t = 170 min). The time behavior of the sensor will be further investigated in follow-up research.
Analysis of State Lifetimes. BPM is a single-molecule technique that allows for investigations of lifetimes of bound and unbound states by analyzing state durations between consecutive switching events. The switching events were determined using the maximum-likelihood multiple-windows change point detection method (MM-CPD). 28 Particles were classified as bound unless the standard deviation of their x−y positions exceeded 50 nm over the duration between two switching events. The characteristic duration of antibody− analogue bonds was determined by analyzing distributions of bound-state lifetimes at different conditions, as shown in Figure 3A. First, the bound-state lifetimes in the absence of analogues were analyzed to characterize the background. The cumulative distribution function (CDF) of these states follows . These short-lived states represent the background signal of the experimental system, caused by a combination of nonspecific interactions and misidentified events by the algorithm. The latter relates to the finite MM-CPD window size (set to 0.3−15 s), which was used to identify switching events. Next, the characteristic duration of the single-molecular bonds was determined by analyzing the bound-state lifetimes in the presence of the analogue. To distinguish between background and specific bound-state lifetimes, the cumulative distribution was fitted using a double-exponential function (CDF = f 1 ·e −t/τ bg + f 2 ·e −t/τ bound , with f 1 + f 2 = 1). The fits reveal two populations of states, with mean lifetimes of ∼4 s and mean lifetimes of around 30 s, respectively, with the fraction of long-lived bound states (f 2 ) increasing for decreasing cortisol concentrations. The data shows that the mean bound-state lifetimes are independent of the cortisol concentration, which indicates that the sensor forms the same type of bonds over the complete concentration range. By controlling the density of binders on the particle and on the substrate, a sensor was developed that is dominated by single-molecule interactions (analogue-antibody) and not by multivalent bonds between particles and substrate (see Figure   S4). The measured bound-state lifetimes correspond to an effective dissociation rate constant of specific bonds on the order of 0.03 s −1 . Association processes between the particle and substrate are reflected in the unbound-state lifetimes. Unbound-state lifetimes are defined as the time separation between two bound states and depend on assay conditions such as analogue and antibody density. Particles that remain in the unbound state during the whole measurement duration are not included in the analysis. In the experiments, the number of particles that contribute to the unbound-state lifetimes varied from about 1000 particles for low cortisol concentration to about 500 for the highest cortisol concentration. The cumulative distribution of the unbound-state lifetimes was fitted using a multiexponential decay function, as described by Lubken et al. 26,29 The multiexponential fitting relates to interparticle heterogeneities of binder densities, in agreement with the fact that measurements on individual particles give single-exponential decay curves; see Figure S4. The obtained mean unbound-state lifetimes increase for higher cortisol concentrations, which is attributed to the higher occupancy of antibody binding sites by cortisol.   Figure 3D−F shows the lifetime analysis of the BPM sensor for cortisol in blood plasma that was filtered with a 50 kDa molecular-weight cutoff. The results in plasma and in the buffer are very similar, for the background, bound-, and unbound-state lifetimes, demonstrating that the biomolecular affinities and nonspecific interactions are hardly affected by filtered plasma.
Continuous Monitoring of Alternating Cortisol Concentrations in Filtered Blood Plasma. The sensor reversibility and sensor response time were investigated by exposing the sensor to series of large fluctuations of cortisol concentrations, in nanomolar and micromolar ranges ( Figure  4). The experiments show that the BPM sensor responds reversibly to increases as well as decreases of cortisol concentrations, with a response time below 15 min, in buffer as well as in filtered blood plasma. The sensors in Figure 4 show blank signals that remain stable within about 10% during the total experiment, as is also seen in Figure 2. However, the absolute value of the signal at zero cortisol concentration differs between individual sensors, as can be seen in Figures 2  and 4. We attribute the differences to variabilities in the surface preparations, causing different areal densities of analogue molecules on the substrate. To compare experiments with different sensors, the data can be plotted with normalized signal values, as shown in Figure 4C. This comparison highlights that experiments in buffer and in blood plasma show sigmoidal curves with strong similarity.
BPM Measurement with Microdialysis Samples from Plasma. Microdialysis was selected as a continuous plasma sampling technique because cortisol is a small molecule that can pass through nanofiltration membranes (Table S1) and microdialysis probes are commercially available for use in future clinical applications. 30−32 Sampling was done from reconstituted lyophilized human blood plasma, spiked with cortisol, and maintained at a temperature of 37°C. Figure 5A illustrates the principles of a microdialysis probe, with analyte molecules diffusing into a perfusing fluid. The perfusion liquid flowed at a speed of 2 μL per min, the dialysate fluid was collected, and BPM measurements were performed in the dialysate. The BPM sensor signal shows a clear response to increases as well as decreases in cortisol concentration, which demonstrates the feasibility of combining the sampling of cortisol from blood plasma using microdialysis, with measurements on the continuous BPM sensor.
One parameter to characterize microdialysis sampling is the recovery, i.e., the ratio between the concentration of the analyte in the sample and the concentration of the analyte in the dialysate: . Recovery values depend on the analyte, membrane, perfusate, and flow-rate properties. We analyzed different experiments and estimated recoveries in the range between 5 and 35% ( Figure S5). Future research will focus on further characterization of recovery and sensing properties for different flows, fluids, probes, and BPM sensor designs.

■ CONCLUSIONS AND OUTLOOK
The goal of this research was to develop a sensor to enable continuous cortisol monitoring in blood plasma and investigate the influence of plasma on the sensing parameters. A BPM sensor was developed with sensitivity in the high nanomolar to low micromolar range, suited for continuous cortisol monitoring over multiple hours in filtered human blood plasma. The reversibility of the sensor was demonstrated by applying alternating cortisol concentrations with increases as well as decreases in cortisol concentration. Reversibility is important for continuous monitoring applications and has been a limitation of cortisol sensors reported in the literature. 20,21,33 Continuous cortisol monitoring in plasma is useful for mapping stress profiles and inflammatory responses of individual patients. Sampling of blood plasma with a microdialysis probe was demonstrated as an important step toward future real-time patient monitoring. In further research, we will investigate the integration of the BPM sensor with microdialysis sampling, including automated calibrations. BPM as a method for continuous biomolecular sensing 24,27 and microdialysis as a method for continuous sampling from complex biological fluids 30−32 are both suited for a wide range of molecules and sample fluids. Therefore, we expect that the combination of BPM and microdialysis will lead to flexible bioanalytical systems for diverse applications in fundamental biological research and patient monitoring.

Materials.
The oligonucleotides used in the study were purchased from IDT. Chemicals used in the study were purchased from Sigma, except if stated otherwise. Custom-made fluid cell stickers (Custom 6 Well Secure Seal) were obtained from Grace Biolabs.
Amine-modified DNA was diluted to 10 μM in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (50 mM MOPS (Sigma-Aldrich; M1254) and 0.5 M NaCl, pH 8.0), of which 72 μL was added to the mixture and left to react for 16 h (room temperature, 850 rpm). A fresh reaction mixture of cortisol, HOBt, EDC, and DIPEA was prepared as before, incubated for 15 min, added to the amine−DNA mixture, and left to react for 6 h. The reaction was quenched by adding 25 μL of 500 mM NH 4 OAc (Sigma-Aldrich; A1542).
The reaction mixture containing cortisol−DNA was dissolved in 0.15 mM NaCl in 98% ethanol, stored at −20°C for 16 h, followed by spinning down at 17,000g for 15 min at 4°C. The pellet was washed a second time (0.15 mM NaCl in 98% ethanol), incubated at −20°C for 75 min, centrifuged, and washed with 70% ethanol. After incubation at −20°C for 75 min, it was centrifuged, and the cortisol− DNA was obtained after lyophilization. The cortisol−DNA was dissolved to 25 μM, and the conjugation was verified using gel electrophoresis with a 15% urea gel at 150 V for 90 min.
Plasma and Cortisol Preparation. Human blood plasma (Sigma P9523-5 mL) was reconstituted using 5 mL of Milli-Q. The 50 kDa filtered plasma was prepared by mixing it with 5 M NaCl/PBS to reach 0.5 M NaCl/plasma, which was filtered using a 50 kDa molecular-weight cutoff centrifugal filter (UFC905008, Millipore) according to the supplier's protocol (total centrifugation time of 20 min).
Cortisol stock was prepared by dissolving 1 mg/mL in methanol (technical grade) and diluted further in either 0.5 M NaCl/PBS, 50 kDa filtered plasma with 0.5 M NaCl, or full plasma, with concentrations ranging from 30 μM down to 123 nM.
Sensor Assembly and Cortisol Detection. On the day of use, 250 μL of functionalized particles was injected (Harvard pump 11 Elite, 40 μL/min withdrawal speed) into the fluid cell (Grace BioLabs). Particles were incubated for 5 min to allow particles to sediment to the substrate and attach to the DNA tethers. Thereafter, the slide was reversed to allow untethered particles to sediment away from the functionalized surface. Second, 400 μL of 100 μM of 1 kDa mPEG-biotin (PG1-BN-1k, Nanocs) was added, which was incubated for 30 min. During incubation, the tethered particles were measured to determine the background signal. Activation Image Recording and Data Analysis. Tethered particles were tracked before, during, and after analogue binding, and after each concentration change, on a Leica Microscope (DMI5000 M with a CTR6000 light source), at a total magnification of 10× using a highspeed FLIR CMOS camera (Point Grey Research Grashopper3 GS3-U3-23S6M, 1920 × 1200, pixel format: 8 raw, Gain 10). The particle motion in a field of view of 1129 × 706 μm 2 was recorded for 5−10 min at the frame rate of 30 Hz with 5 ms exposure time under darkfield illumination conditions. The localization of particles was done using phasor-based localization. 35 The analysis of particle motion and detection of switching events were done using the maximumlikelihood multiple-windows change point detection method (MM-CPD) 28 and the method described in an earlier publication. 23